Clinical StudyStudy of Demarcation Line Depth in Transepithelial versusEpithelium-Off Accelerated Cross-Linking (AXL) in Keratoconus

Yehia Salah, Kholoud Omar, Ahmed Sherif , and Sarah Azzam

Department of Ophthalmology, Cairo University, Giza, Egypt

Correspondence should be addressed to Sarah Azzam; sarahazzam@ymail.com

Received 27 March 2019; Revised 3 July 2019; Accepted 6 September 2019; Published 26 September 2019

Academic Editor: Stefano Baiocchi

Copyright © 2019 Yehia Salah et al. +is is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Aim. +is is a prospective interventional clinical trial to assess the depth of the demarcation line in transepithelial versusepithelium-off accelerated corneal cross-linking (AXL) in keratoconus patients. Methods. +is prospective clinical trial wasconducted on 40 eyes of 20 patients. Each patient had transepithelial AXL in one eye and epithelium-off AXL in the contralateraleye applying UVA light with an irradiance of 45mW/cm2 for 2.4 minutes and 30mW/cm2 for 4 minutes. +e depth of thedemarcation line was measured using anterior segment OCT (Topcon 3D OCT-2000) one month postoperative for both eyes.Results. +e demarcation line was patchy in 50% of the transepithelial AXL eyes, the other half showing a demarcation line at amean depth of 183± 41.6 μm. In the epithelium-off AXL technique, the demarcation line was well defined in all cases with a meandepth of 219± 27.3 μm. +ere was a statistically significant difference in corneal demarcation line depth between transepithelialand epithelium-off techniques (P � 0.008 and P< 0.05).+e shallower demarcation line in the transepithelial group suggests that itis less effective. Conclusion. Based on the depth of the demarcation line, the cross-linking effect of epi-off AXL seems moreefficacious than after transepithelial AXL. +e future will show if the biomechanical effect will be sufficient to stop progression ofkeratoconus similarly after standard CXL. +is trial is registered with NCT04045626.

1. Introduction

Keratoconus is a degenerative corneal disorder associatedwith corneal ectasia and thinning with eventual loss of visualacuity following myopia, irregular astigmatism, and cornealscarring [1]. Corneal collagen cross-linking is a surgicalmodality for increasing the corneal biomechanical strengthto arrest keratoconus progression [2, 3]. Standard cornealcross-linking treatment of progressive keratoconus wasintroduced by Wollensak et al. from Germany in 2003. It issometimes also called the Dresden protocol and includespreoperative removal of the central corneal epithelium,application of the photosensitizer riboflavin, and UVA ir-radiation with an irradiance of 3mW/cm2 for 30 minuteswith a total surface dose of 5.4 J/cm2 [4]. So-calledaccelerated cross-linking (AXL) has been proposed toshorten the duration of the procedure. It is based on theBunsen-Roscoe law of reciprocity increasing the UVA ir-radiance and significantly reducing the irradiation timeresulting in a similar overall UVA dose [5]. Transepithelial

cross-linking, gained ground by leaving epithelium intact,improving patient comfort and minimizing the risk of in-fection associated with epithelial removal. However, its ef-ficacy remained controversial as the epithelial barrier limitsthe depth and amount of cross-linking, as evidenced by ashallow and uneven distribution of the keratocytes apoptosison confocal microscopy and ectatic progression in manyclinical trials as compared to epithelium-off CXL [6]. +estratified corneal epithelium with tight junctions presents asa lipophilic barrier to the absorption of the hydrophilicriboflavin macromolecule. Standard riboflavin formulationswere modified with addition of EDTA, benzalkoniumchloride (BAC), and trometamol, which loosen up the ep-ithelial junctions, enhancing the permeability and riboflavindiffusion across the epithelium and superficial stroma [7].

Studies have shown that the effective depth of standardCXL is confined to the anterior 300 um of the cornea asdemonstrated by the depth of the keratocyte apoptosis withlacunar edema on histology and the clinical demarcation line[5].

HindawiJournal of OphthalmologyVolume 2019, Article ID 3904565, 4 pageshttps://doi.org/10.1155/2019/3904565

+e purpose of our study is to evaluate and compare thedepth of the corneal stromal demarcation line using AS-OCT after transepithelial cross-linking versus acceleratedepi-off cross-linking procedures.

2. Patients and Methods

+is prospective comparative interventional study com-prised patients with progressive keratoconus. +e study wasapproved by the local ethical committee and followed theprinciples of the Declaration of Helsinki. 40 eyes of 20patients were divided into 2 groups: Group A: transepithelialAXL; Group B: epithelium-off AXL. Inclusion criteria wereprogressive keratoconus, grade 1-2 keratoconus (Amsler–Krumeich classification) Snellen best corrected visual acuityof 0.4 or better, and pachymetry of 400 μm or more atthinnest location.

Progressive keratoconus was defined as an increase inkmax of 1.00 diopter [D], an increase of manifest refractioncylinder of 1.0D, or an increase of manifest refractionspherical equivalent of 0.5D over the period of 1 year. CXLtreatment was performed with a KXL I UVA source (AvedroInc., Waltham, MS, USA).

In transepithelial AXL, ParaCel™ (containing 0.25%riboflavin and benzalkonium chloride) was instilled in theform of 1 drop every 1.5 minutes for 4.5 minutes, and thenVibeX Xtra was instilled in the form of 4 drops at 5.5 minutesfollowed by 1 drop at 6.5 minutes and again every 1.5minutes for three times. Total soak time of riboflavin was 11minutes followed by UVA irradiation with an intendedirradiance of 45mW/cm2 (total surface dose of 7.2 J/cm2) for2.4 minutes.

In epithelium-off AXL, the corneal epithelium was re-moved using a blunt spatula (8.0 to 9.0mm in diameter).VibeX Rapid (riboflavin 0.1%) was instilled on the center ofthe cornea every 2 minutes for 10 minutes followed byultraviolet-A irradiation with an intended irradiance of30mW/cm2 (total surface dose of 7.2 J/cm2) for 4 minutes.At the end of the procedure, a bandage contact lens wasplaced over the cornea and removed after full epithelization(postoperative 4-5 days). Postoperative medications in-cluded moxifloxacin 0.5% (Vigamox, Alcon, Inc., Canada) 4times daily for 2 weeks and artificial tears 4 times daily forone month with ascorbic acid for one month. After removalof contact lens, patients received fluoromethelone acetate 4times daily tapered over 2 weeks.

2.1. Evaluation of Depth of the Demarcation Line. Anteriorsegment optical coherence tomography (AS-OCT) (Topcon3D OCT-2000) was performed to evaluate the cornealstroma for the presence of the demarcation line 1 monthpostoperative by an independent observer unaware of thepurpose of this study. +e demarcation line measurementsof this study include the epithelial thickness of about 50 μm.

3. Statistical Analysis

Microsoft EXCEL 2010 and SPSS of Windows software(version 20.0, SPSS Inc.) were used for statistical analysis.

Normality of all data samples was first checked by means ofthe Kolmogorov–Smirnov test. +en t-test was possible, andthe Student t-test for paired data was performed for allparameter comparisons between preoperative and post-operative examinations using the same level of significance(P< 0.05) in all cases. And in comparing the 2 groups weused the unpaired t-test.

4. Results

+is clinical prospective study included 40 eyes of 20 pa-tients; each patient had transepithelial CXL in one eye andepithelium-off CXL in the other. 11 females and 9males wereincluded in the study. +e mean patient age was 32 (rangingfrom 16 to 40 years). +e mean preoperative CCT was484.55± 35.47 μ in the transepithelial group and 477.75±37.06 μ in the epi-off group (P � 0.557).

4.1. Demarcation Line One Month Postoperatively. +e de-marcation line was patchy and ill-defined in 50% of thetransepithelial eyes, the depth of which could not be assessed(Figure 1). +e other half showed a clear demarcation line.However, there was a clear demarcation line in all epithe-lium-off eyes. +ere was a statistically significant differencein the incidence of a patchy corneal demarcation line be-tween transepithelial and epithelium-off techniques(P � 0.001 and P< 0.05).

4.2. Demarcation Line Depth One Month Postoperatively.+emean depth of the corneal demarcation line was 183 μmin the transepithelial technique and 219 μm in the epithe-lium-off technique (Figures 2 and 3).+ere was a statisticallysignificant difference in corneal demarcation line depthbetween transepithelial and epithelium-off techniques(P � 0.008 and P< 0.05; Table 1).

4.3. Postoperative Complications. Grade I corneal haze(graded according to Fantes scoring) was found in 4 eyesonly in transepithelial CXL and in 9 eyes in epithelium-offCXL. No delayed epithelialization or infective keratitis wasseen in either group.

5. Discussion

Corneal CXL with riboflavin and ultraviolet-A (UVA) is atechnique to strengthen corneal tissue [8]. +e cornealepithelium is the critical obstacle to riboflavin permeationinto the corneal stroma [8, 9]. However, the removal ofepithelium can cause some complications such as severepostoperative pain, temporary visual blurring, epithelialhealing problems, haze, viral reactivation, and even cornealmelting. To prevent these serious problems, some re-searchers have performed a number of studies on a specialriboflavin solution, which can penetrate the intact epithe-lium, such as benzalkonium chloride, EDTA, and trome-tamol [7].

In the epi-off group, our results were comparable toMoramarco et al. [10] and Jiang et al. [11] who performed

2 Journal of Ophthalmology

epi-off AXL using pulsed UVA light with 8 minutes of UVAexposure at 30mW/cm2 and energy dose of 7.2 J/cm2. +emean depth of the stromal demarcation line was213± 47.38 μm and 201.64± 27.72 μm, respectively.

Bouheraoua et al. showed similar results using irradianceof 30mW/cm2 for 3 minutes (5.4 J/cm2 surface dose). +ecorneal demarcation line was visible in 87.5% (mean depth184.2± 38.9 μm) in the A-CXL group [12].

+e depth of the demarcation line in our study of epi-thelium-off cases was found to be shallower (mean 219 μ)than in other studies using different accelerated protocols. Ina study by Kymionis et al., patients were treated for 10minutes in an intended irradiance of 9.0mW/cm2 corre-sponding to a total surface dose of 5.4 J/cm2.+emean depthof the corneal stromal demarcation line was288.46± 42.37mm [13]. Another study by Kymionis et al.,using high intensity CXL protocol of 18mW/cm2 UVAirradiation for 7 minutes, the mean corneal stromal de-marcation line depth was 313.37± 48.85 um [14].

Similarly, Lhuillier et al. using an accelerated protocol of10min, 9mW/cm2 ultraviolet-A, corresponding to a totalsurface dose of 5.4 J/cm2 reported a mean depth of thestromal demarcation line of 331.2± 62.7 μm [15].

Our results on AXL were markedly different from Yamet al. who used a classic protocol of 3.0mW/cm2 for 30minutes (surface dose 5.4 J/cm2). +e median CXL de-marcation line depth at the central cornea was 323.0± 48.6,suggesting a significantly weaker cross-linking effect of AXLcompared to standard CXL [5].

As regards for the transepithelial AXL technique, UVAirradiation was performed for 2.4 minutes, with anintended power of 45mW/cm2, giving a total energy of7.2 J/cm2. 50% of the cases had a patchy demarcation lineand 50% had a demarcation line at a mean depth of183 μm.

+ese results were similar to Artola et al. who reported ademarcation line depth between 153 and 230 μm, with amean value of 205.19 μm [16].

Figure 3: A corneal demarcation line at a central depth of 227 μm after epithelium-off cross-linking.

Table 1: Summary of results.

Transepithelial, mean SD Accelerated epithelium-off, mean SD P valueCentral corneal thickness 484.55 35.47 477.75 37.06 0.557Demarcation line depth 183.0 41.60 219.30 27.30 0.008

Figure 1: A patchy corneal demarcation line (white arrow) after transepithelial cross-linking.

Figure 2: A barely visible corneal demarcation line at a central depth of 185 μm after transepithelial cross-linking.

Journal of Ophthalmology 3

Our study’s findings support the review by Liu et al. whocollected eligible studies retrieved from four electronic da-tabases, including CENTRAL, ClinicalTrials.gov, PupMed,and Ovid MEDLINE. 24 comparative studies on trans-epithelial cross-linking compared with standard cross-linking were included and pooled for analysis. +e de-marcation line of standard CXL was significantly deeperthan that of transepithelial cross-linking; the mean differ-ence of the depth of the demarcation line (DDL) was133.49 μm [17].

Both transepithelial and epi-off AXL have been shown tobe biomechanically less efficacious than standard CXL [5].+e weaker effect is also reflected by the present study withthe difference in the DL depth and the often patchy andirregular configuration or even absence of the DL intransepithelial AXL probably due to an inhomogenousdistribution of the photosensitizer riboflavin [18].

In conclusion, we found that the demarcation line wasnot consistent in transepithelial cases, and it was found to bemore superficial than in standard CXL as has also beenfound by other studies with different AXL protocols. Ashallower demarcation line demonstrates a weaker cross-linking effect. It has been shown that the CXL effect has anatural saturation effect which is maximally high in a specifictissue layer. +e effect can only be increased further byincreasing the volume of cross-linked tissue which is re-flected and revealed by the greater depth of DL [5].

+e future will show if the clearly weaker cross-linkingeffect of epi-off and transepithelial AXL compared tostandard CXL will be sufficient to stop progression ofkeratectasia.

Data Availability

+e data used to support the findings of this study areavailable from the corresponding author upon request.

Conflicts of Interest

+e authors declare that they have no conflicts of interest.

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